Postbiotic extract and preparation process and use thereof

ABSTRACT

Disclosed herein is a process for producing a postbiotic extract, which includes providing a first material having a first isoelectric point ranging from pH 1 to pH 6 and a second material having a second isoelectric point ranging from pH 4 to pH 8, admixing the first material and a probiotic microorganism with water having a pH greater than the second isoelectric point, so as to form a mixture, adding the second material into the mixture and then adjusting a pH of the second material-added mixture to between the first and second isoelectric points so that a precipitate is formed, and subjecting the precipitate to a cell wall isolation treatment to obtain the postbiotic extract. Use of the postbiotic extract is also disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. patent applicationSer. No. 17/037,090, filed on Sep. 29, 2020. The entire content of theprior application is incorporated herein by reference.

FIELD

The present disclosure relates to a postbiotic extract and a process ofpreparing the same. The present disclosure also relates to use of thispostbiotic extract to improve gut health and to inhibit biofilmformation.

BACKGROUND

Probiotics are resident normal flora of the intestinal tract andbelieved to play important roles in regulating proper intestinalimmunity and digestion by balancing intestinal microflora. Thesebeneficial microorganisms are widely used as live microbial dietarysupplement and can help restoring intestinal microfloral balance. Manyspecies of lactic acid bacteria (LAB), such as Lactobacillus spp.,Lactococcus spp., Streptococcus spp., Enterococcus spp., andBifidobacterium spp., are generally considered as probiotics.Furthermore, some of Bacillus spp. and some yeasts and Saccharomycesspp. have also been found as suitable candidates.

Probiotics are viable by definition, and their stability and viabilityare considered to be crucial for their health benefits. Paraprobioticsor postbiotics have emerged to denote that non-viable microbial cells,microbial fractions, or cell lysates might also offer physiologicalbenefits to the host by providing additional bioactivity. Postbioticefficacy is based on the microbial metabolites, proteins, lipids,carbohydrates, vitamins, organic acids, cell wall components, or othercomplex molecules that are generated in the matrix that is fermented.These postbiotics have drawn attention because of their clear chemicalstructure, safety dose parameters, long shelf life and the content ofvarious signaling molecules which may have anti-inflammatory,immunomodulatory, anti-obesogenic, antihypertensive,hypocholesterolemic, anti-proliferative, and antioxidant activities.

Postbiotics can be obtained using cell disruption methods, which includeheat, enzymatic treatment, solvent extraction, and sonication. However,these methods usually have low extraction efficiency and use largeamounts of solvents.

Thus, there is still a need to develop a new method for efficientlyproducing a postbiotic from a probiotic microorganism.

SUMMARY

Accordingly, in a first aspect, the present disclosure provides aprocess for producing a postbiotic extract, including the steps of:

-   -   a) providing a first material having a first isoelectric point        ranging from pH 1 to pH 6, and a second material having a second        isoelectric point ranging from pH 4 to pH 8, wherein the second        isoelectric point is greater than the first isoelectric point,        and the first isoelectric point and the second isoelectric point        have a pH difference ranging from 0.5 and 3;    -   b) admixing the first material and a probiotic microorganism        with water having a pH value greater than the second isoelectric        point, so as to form a mixture;    -   c) adding the second material into the mixture, followed by        adjusting a pH value of the second material-added mixture to        between the first isoelectric point and the second isoelectric        point, so that a precipitate is formed; and    -   d) subjecting the precipitate to a cell wall extraction        treatment to obtain the postbiotic extract.

In a second aspect, the present disclosure provides a postbiotic extractwhich is prepared by a process as described above.

In a third aspect, the present disclosure provides a food productincluding a postbiotic extract as described above.

Ina fourth aspect, the present disclosure provides a method forinhibiting biofilm formation, which includes applying a postbioticextract as described above onto an object.

In a fifth aspect, the present disclosure provides a method forimproving gut health, which includes administering to a subject apostbiotic extract as described above.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and advantages of the presentdisclosure will become apparent with reference to the following detaileddescription and the exemplary embodiments taken in conjunction with theaccompanying drawings, in which:

FIG. 1 is a digital image showing a result of sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis ofpostbiotic extracts of Example 1, infra.

DETAILED DESCRIPTION

It is to be understood that, if any prior art publication is referred toherein, such reference does not constitute an admission that thepublication forms a part of the common general knowledge in the art, inTaiwan or any other country.

For the purpose of this specification, it will be clearly understoodthat the word “comprising” means “including but not limited to”, andthat the word “comprises” has a corresponding meaning.

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich the present disclosure belongs. One skilled in the art willrecognize many methods and materials similar or equivalent to thosedescribed herein, which could be used in the practice of the presentdisclosure. Indeed, the present disclosure is in no way limited to themethods and materials described.

The present disclosure provides a process for producing a postbioticextract, comprising the steps of:

-   -   a) providing a first material having a first isoelectric point        ranging from pH 1 to pH 6, and a second material having a second        isoelectric point ranging from pH 4 to pH 8, wherein the second        isoelectric point is greater than the first isoelectric point,        and the first isoelectric point and the second isoelectric point        have a pH difference ranging from 0.5 and 3;    -   b) admixing the first material and a probiotic microorganism        with water having a pH value greater than the second isoelectric        point, so as to form a mixture;    -   c) adding the second material into the mixture, followed by        adjusting a pH value of the second material-added mixture to        between the first isoelectric point and the second isoelectric        point, so that a precipitate is formed; and    -   d) subjecting the precipitate to a cell wall extraction        treatment to obtain the postbiotic extract.

According to the present disclosure, the probiotic microorganism may beselected from the group consisting of Bacillus spp., Streptococcus spp.,Lactococcus spp., Abiotrophia spp., Aerococcus spp., Carnobacteriumspp., Enterococcus spp., Lactobacillus spp., Leuconostoc spp.,Oenococcus spp., Pediococcus spp., Tetragenococcus spp., Vagococcusspp., Weissella spp., Bifidobacterium spp., Saccharomyces spp.,Kluyveromyces spp., Staphylococcus spp., Pediococcus spp.,Propionibacterium spp., and combinations thereof.

According to the present disclosure, the Lactobacillus spp. may beselected from the group consisting of Lactobacillus plantarum,Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus rhamnosus,Lactobacillus paracasei, and combinations thereof.

According to the present disclosure, the Bifidobacterium spp. may beselected from the group consisting of Bifidobacterium bifidum,Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium breve,Bifidobacterium animalis, and combinations thereof.

According to the present disclosure, the Bacillus spp. may be selectedfrom the group consisting of Bacillus coagulans, Bacillus subtilis,Bacillus clausii, and combinations thereof.

According to the present disclosure, the probiotic microorganism may bealive or dead, concentrated or non-concentrated, or in the form of aliquid, a paste, a semi-solid, or a solid (e.g. a pellet, a granule, ora powder), and may be heat-inactivated, frozen, dried, or freeze-dried(for example, may be in a freeze-dried form or a spray/fluid bed driedform). In an exemplary embodiment, the probiotic microorganism isheat-inactivated and is in a spray-dried powder form.

According to the present disclosure, the heat inactivation of theprobiotic microorganism may be conducted at 60° C. to 140° C. for 1second to 30 minutes. In an exemplary embodiment, the heat inactivationis conducted at 73±2° C. for 15 seconds.

According to the present disclosure, the first material may be selectedfrom the group consisting of nonfat dry milk, casein, whey proteins,soybean proteins, pea proteins, egg proteins, rice proteins, hydrolyzedproteins, corn proteins, wheat proteins, barley proteins, gelatin,collagen, amino acids (for instance, branched chain amino acids),chitosan, chitin, and combinations thereof. In an exemplary embodiment,the first material is a whey protein.

According to the present disclosure, the second material may be selectedfrom the group consisting of sodium alginate, agar, carrageenan, pectin,arabic gum, xanthan gum, locust bean gum, starch (such as modifiedstarch), trehalose, dextrin (such as resistant maltodextrin), syrup,guar gum, konjac powder, vegetable fiber, synthetic fiber,semi-synthetic fiber, and combinations thereof. In an exemplaryembodiment, the second material is dextrin.

In an exemplary embodiment of the present disclosure, the firstisoelectric point and the second isoelectric point have a pH differenceof 0.8.

According to the present disclosure, the precipitate may be obtained bymethods well known in the art, including but not limited tocentrifugation, filtration, and gravity settling. In an exemplaryembodiment, the precipitate is obtained by filtration.

As used herein, the terms “isolation treatment” and “extractiontreatment” can be used interchangeably, and mean that a cell wallcomponent or a microbial metabolite is to be separated from a cell wall,in which it was originally present, through a treatment.

According to the present disclosure, the procedures and conditions ofthe cell wall isolation treatment are within the expertise and routineskills of those skilled in the art (for example, see Pei-Jun Tian et al.(2015), Int. J. Mol. Sci., 16(8): 20033-20049).

The present disclosure also provides a postbiotic extract which isprepared by a process as described above.

The present disclosure also provides a food product, which includes apostbiotic extract as described above. According to the presentdisclosure, for preparing the food product, the postbiotic extract maybe incorporated into an edible material using a standard technique wellknown to one of ordinary skill in the art. For instance, the aforesaidpostbiotic extract may be directly added to the edible material, or maybe utilized for preparing an intermediate composition (e.g., a foodadditive or a premix) suitable to be subsequently added to the ediblematerial.

According to the present disclosure, the food product may be in the formof fermented foods, processed foods, health foods, or dietarysupplements.

The food product according to the present disclosure may further includeat least one probiotic microbe. As used herein, the terms “probioticmicrobe” and “probiotic” are used interchangeably, and refer topreparations microorganisms of live microorganisms. These microorganismsmay remain and survive in the gastrointestinal tract after ingested byan animal, and can exert a desired effect (e.g. gut microbiota modifyingeffect, preventive or therapeutic effect, etc.).

Probiotic microorganisms suitable for use in the present disclosureinclude, but are not limited to, a Lactobacillus sp., a Enterococcussp., a Streptococcus sp., a Pediococcus sp., a Bacillus sp., aBifidobacterium sp., yeasts, and their combinations.

According to the present disclosure, the food product may furthercomprise an additional food additive selected from the group consistingof starch, dextrin, lactose, maize flour, rice flour, tricalciumphosphate, silicon dioxide, magnesium stearate, calcium carbonate,glucose, sucrose, fructose, sugar alcohol, oligosaccharides, sugarsubstitutes, fruit juice powder, yeast powder, nonfat dry milk, casein,whey proteins, amino acids, citric acid, citrate, lactic acid, lactate,nucleotides, and their combinations.

In addition, the postbiotic extract according to the present inventionmay be prepared in the form of a pharmaceutical composition.

According to the present disclosure, the pharmaceutical composition maybe formulated into a suitable dosage form for parenteral, oral ortopical administration using technology well known to those skilled inthe art. The suitable dosage form includes, but is not limited to,sterile powder, tablets, troches, lozenges, pellets, capsules,dispersible powder or granules, solutions, suspensions, emulsions,syrup, elixir, slurry, external preparations, and the like.

The pharmaceutical composition according to the present disclosure mayfurther include a pharmaceutically acceptable carrier widely employed inthe art of drug-manufacturing. For instance, the pharmaceuticallyacceptable carrier may include one or more of the following agents:solvents, buffers, suspending agents, decomposers, disintegratingagents, dispersing agents, binding agents, excipients, stabilizingagents, chelating agents, diluents, gelling agents, preservatives,lubricants, absorption delaying agents, liposomes, and the like. Thechoice and amount of the aforesaid agents are within the expertise androutine skills of those skilled in the art.

The present disclosure also provides a method for inhibiting biofilmformation, which includes applying a postbiotic extract as describedabove onto an object.

As used herein, the term “biofilm formation” refers to the attachment ofmicroorganisms to surfaces and the subsequent development of multiplelayers of cells.

As used herein, the term “inhibition” or “inhibiting” refers to adecrease of biofilm associated microorganism formation and/or growth.The microorganisms may include gram-positive or gram-negative bacteria,yeasts, and fungi.

According to the present disclosure, the object may be a medical device,a medical instrument, a dressing, a bandage, a food preparation surface,a food packaging surface, a manufacturing surface, a consumer good, awater treatment system, a water delivery system, or a ventilationsystem.

In certain embodiments, the object may be selected from the groupconsisting of a denture, a mouth guard, a dairy line, a water line, anadhesive bandage, a component of an HVAC (heating, ventilation, andair-conditioning) system, a component of a water treatment facility, acomponent of a vacuum or a vacuum cleaner, a vacuum cleaner bag, avacuum cleaner filter, an air filter, a component of a cooling tower, atoy, a window, a door, a window frame, a doorframe, a medicalinstrument, a dental instrument, a bathroom tile, a kitchen tile, foodindustry processing instruments, hospital tables and beds, an animalwater dish, a washing machine, a dish washer, a towel, a dish, a bowl, autensil, a cup, a glass, a cutting board, a dish drying tray, awhirlpool bathtub, a sink, a toilet, a toilet seat, a swimming pool, abirdbath, a planter, a garden hose, a fish pond, an oil pipe, a gaspipe, a dairy line filter, a line used in food and beveragemanufacturing, a cosmetic container, an outdoor pond liner, a tap andwater spout, a humidifier, a humidifier filter, a bathroom tile, abathroom fixture, a toilet lid, a swimming pool liner, a swimming poolskimmer, a swimming pool filter, a hot tub line, a hot tub filter, awashing machine liner, a dishwasher liner, an animal water dish, a foodstorage container, a beverage storage container, a plate, a cup, a fork,a knife, a spoon, a garbage bag, and a counter top.

According to the present disclosure, biofilm formation may be caused bya microbe selected from the group consisting of Campylobacter spp.,Clostridium perfringens, Escherichia coli, Listeria monocytogenes,Vibrio cholerae, Salmonella spp., Staphylococcus spp., and combinationsthereof.

Examples of the Staphylococcus spp. include, but are not limited to,Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcusagalactiae, Staphylococcus saprophyticus, Staphylococcus haemolyticus,Staphylococcus warneri, Staphylococcus hominis, Staphylococcus simulans,Staphylococcus lugdunensis, Staphylococcus schleiferi, Staphylococcuscapitis, Staphylococcus caprae, Staphylococcus pasteuri, Staphylococcuscohnii, Staphylococcus xylosus, and Staphylococcus saccharolyticus.

The present disclosure also provides a method for improving gut health,which includes administering to a subject a postbiotic extract asdescribed above.

As used herein, the term “improving gut health” means that anindividual, upon treatment with the postbiotic extract, exhibits ahealthy gut/intestinal microbiota, which is beneficial for human oranimal health and suitable for a maintenance and/or an improvement ofthe digestion of said individual. Such a healthy gut/intestinalmicrobiota is ultimately linked to proper nutrient absorption, adequategrowth, less colic, less infection, less diarrhea, and the best guthealth.

The disclosure will be further described by way of the followingexamples. However, it should be understood that the following examplesare solely intended for the purpose of illustration and should not beconstrued as limiting the disclosure in practice.

EXAMPLES General Experimental Materials:

-   -   1. The probiotics used in the following experiments are listed        in Table 1.

TABLE 1 Bacteria Strain Source Lactobacillus spp. Lactobacillusplantarum CB102 Department of Lactobacillus acidophilus JCM1132 FoodScience Lactobacillus casei JCM1134 and Biotechnology, National ChungHsing Bifidobacterium spp. Bifidobacterium bifidum JCM1255 University,Bifidobacterium lactis JCM10602 Taiwan Bifidobacterium longum CB108Bacillus spp. Bacillus coagulans CB106

-   -   2. Human colon adenocarcinoma cell line Caco-2 was purchased        from the Bioresource Collection and Research Center of the Food        Industry Research and Development Institute (BCRC of FIRDI,        Taiwan). Caco-2 cells were grown in a 10-cm Petri dish        containing Dulbecco's Modified Eagle's Medium (DMEM) (Thermo        Fisher Scientific) supplemented with 10% fetal bovine serum        (FBS). The Caco-2 cells were cultivated in an incubator with        culture conditions set at 37° C. and 5% CO₂. Medium change was        performed every two to three days. Cell passage was performed        when the cultured cells reached 80%-90% of confluence.

General Procedures:

-   -   1. Determination of transforming growth factor-(3 (TGF-β)        content

The TGF-β content was determined using an enzyme-linked immunosorbentassay (ELISA) kit (Cat. No. 559119, BD Biosciences) in accordance withthe manufacturer's instructions.

Example 1. Preparation of Postbiotic Extract of Present Disclosure

A respective one of Lactobacillus plantarum CB102 (deposited at theDeutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH(Inhoffenstraße 7B, 38124 Braunschweig, Germany) under an accessionnumber DSM 33894 on 6 Sep. 2021 in accordance with the Budapest Treaty),Lactobacillus acidophilus JCM1132, Lactobacillus casei JCM1134,Bifidobacterium bifidum JCM1255, Bifidobacterium lactis JCM10602, andBifidobacterium longum CB108 (deposited at the Deutsche Sammlung vonMikroorganismen und Zellkulturen (DSMZ) GmbH (Inhoffenstraße 7B, 38124Braunschweig, Germany) under an accession number DSM 33895 on 6 Sep.2021 in accordance with the Budapest Treaty) was inoculated in aLactobacilli MRS broth (BD DIFCO, Cat. No. DF0881-17-5), and was thencultivated in an incubator (37° C., 5% CO₂) for 16 hours. The respectiveresultant culture was inactivated by high-temperature short-time (HTST)pasteurization (73±2° C., 15 seconds), followed by centrifugation at10,000 rpm and 25° C. for 15 minutes. The resultant cell pellet wascollected, followed by spray-drying. A portion of the respective driedbacterial cell powder was subjected to a pretreatment as follows.

A suitable amount of whey protein having an isoelectric point of 4.4(NZMP, Cat. No. WPC80) was dissolved in water, and the resultant 10%whey protein solution (w/v, g/L) was adjusted to pH 7.5 through additionof sodium carbonate, followed by adding a suitable amount of arespective one of the six dried bacterial cell powders obtained aboveunder agitation to reach a final concentration of 5% (w/v, g/L).Thereafter, dextrin having an isoelectric point of 5.2 (ZHUCHENGDONGXIAO, Cat. No. Maltodextrin DE8-10) was slowly added into theresultant mixture to reach a final concentration of 6% (w/v, g/L),followed by adjusting the dextrin-added mixture to a pH of 4.8 usinglactic acid, so that a precipitate was formed due to chargeneutralization. Filtration was conducted using a filter paper having apore size of 25 μm, so as to obtain the precipitate. The precipitate wassubjected to a spray drying treatment, thereby obtaining a pretreatedbacterial cell powder.

The isolation and extraction of the cell wall of each pretreatedbacterial cell powder was conducted using a method slightly modifiedfrom that described by Pei-Jun Tian et al. (2015), Int. J. Mel. Sci., 16(8): 20033-20049. Briefly, 50 mg of the respective pretreated bacterialcell powder was mixed with 1 mL of 10% lactic acid, followed by heatingin a water bath incubator (80° C.) for 60 minutes. After centrifugationat 10,000 g for 15 minutes, the resultant pellet was collected and wasmixed with 1 mL of a solution containing a 0.5 M citrate solution andethanol (4:10, v/v, pH 4.6), followed by incubation overnight. Aftercentrifugation at 10,000 g for 20 minutes, the pellet thus obtained waswashed with 95% ethanol, followed by heating in a dry bath incubator(80° C.) for 40 minutes to remove ethanol. Thus, a postbiotic extractwas obtained. The resultant postbiotic extract was used for thefollowing example and is referred to as “the postbiotic extract of thepresent disclosure” hereinafter.

In addition, for the sake of comparison, another portion of therespective one of the six dried bacterial cell powders, which was notpretreated according to the procedures described above, was subjected tothe same isolation and extraction processes. The postbiotic extract thusobtained was used for the following example and is referred to as “thepostbiotic extract of the prior art” hereinafter.

Example 2. Analysis of Extraction Yield and Protein Content

In order to determine the extraction yield and the protein content, thepostbiotic extract of the present disclosure and the postbiotic extractof the prior art obtained in Example 1 were subjected to the followinganalyses.

A. Determination of Extraction Yield

The weight of each of the twelve postbiotic extracts was recorded. Theextraction yield (%) of the respective postbiotic extract was calculatedusing the following Equation (I):

A==(B/50)×100  (I)

where A==extraction yield (%)

-   -   B=weight of respective postbiotic extract (mg)

The result is shown in Table 2 below. It can be seen from Table 2 thatthe extraction yields of the six postbiotic extracts of the presentdisclosure were significantly higher than those of the six postbioticextracts of the prior art, indicating that the process of the presentdisclosure can effectively produce postbiotic extracts from probiotics.

TABLE 2 Extraction yield (%) The postbiotic extract of The postbioticextract Strain the present disclosure of the prior art Lactobacillus43.34% 21.40% plantarum CB102 Lactobacillus 44.14% 20.54% acidophilusJCM1132 Lactobacillus casei 43.94% 21.11% JCM1134 Bifidobacterium 30.97%15.44% bifidum JCM1255 Bifidobacterium 31.24% 15.83% lactis JCM10602Bifidobacterium 31.82% 16.75% longum CB108

B. Determination of Protein Content

The respective postbiotic extract was dissolved in a phosphate buffersolution (containing 8 g/L NaCl, 0.2 g/L KCl, 1.44 g/L Na₂HPO₄, and 0.24g/L KH₂PO₄, and having a pH of 6.2), followed by determining the proteincontent with Pierce™ BCA Protein Assay Kit (Thermo Scientific, Cat. No.23225) according to the manufacturer's instructions.

The result is shown in Table 3 below. It can be seen from Table 3 thatthe protein contents of the six postbiotic extracts of the presentdisclosure were significantly higher than those of the six postbioticextracts of the prior art.

TABLE 3 Protein content The postbiotic extract of The postbiotic extractStrain the present disclosure of the prior art Lactobacillus 0.39% 0.20%plantarum CB102 Lactobacillus 0.41% 0.21% acidophilus JCM1132Lactobacillus casei 0.40% 0.20% JCM1134 Bifidobacterium 1.40% 0.69%bifidum JCM1255 Bifidobacterium 1.38% 0.67% lactis JCM10602Bifidobacterium 1.35% 0.70% longum CB108

C. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)Analysis

1 g of the respective postbiotic extract was dissolved in 20 mL ofwater, followed by performing SDS-PAGE analysis using an electrophoresissystem (Bio-Rad).

Referring to FIG. 1 , the band intensities of the six postbioticextracts of the present disclosure were significantly higher than thoseof the six postbiotic extracts of the prior art. The results of thisexample indicate that the process of the present disclosure is effectivein producing a postbiotic extract from bacterial cells, and theresultant postbiotic extract contains high levels of cell wallcomponents (such as peptidoglycan, lipoteichoic acid, teichoic acid,glycoprotein, and proteoglycan).

Example 3. Evaluation for Inhibition Effect of Postbiotic ExtractAccording to this Disclosure on Biofilm Formation of Staphylococcusaureus Experimental Procedures:

Staphylococcus aureus was inoculated into a tryptic soy broth (TSB) (BDBacto™, Cat. No. DF0370-17-3), followed by cultivation in an incubator(37° C., 5% CO₂) for 16 hours. The resultant culture was divided into 17groups, including one control group, eight comparative groups (i.e.,comparative groups L-1 to L-4 and B-1 to B-4), and eight experimentalgroups (i.e., experimental groups L-1 to L-4 and B-1 to B-4). Theculture of each group was incubated in a respective well of a 96-wellculture plate at a cell number of 1×10¹⁰ CFU/L. Thereafter, each of thecultures of the eight comparative groups and eight experimental groupswas treated with the respective test postbiotic extract so that theculture of each group had a final concentration of the respective testpostbiotic extract as shown in Table 4. The culture of the control groupreceived no treatment.

TABLE 4 Final concentration Group Test postbiotic extract (mg/L) Controlgroup — 0 Comparative group L-1 The postbiotic extract of the 25Comparative group L-2 prior art from Lactobacillus 50 Comparative groupL-3 plantarum CB102 100 Comparative group L-4 200 Experimental group L-1The postbiotic extract of the 25 Experimental group L-2 presentdisclosure from 50 Experimental group L-3 Lactobacillus plantarum CB102100 Experimental group L-4 200 Comparative group B-1 The postbioticextract of the 25 Comparative group B-2 prior art from Bifidobacterium50 Comparative group B-3 longum CB108 100 Comparative group B-4 200Experimental group B-1 The postbiotic extract of the 25 Experimentalgroup B-2 present disclosure from 50 Experimental group B-3Bifidobacterium longum CB108 100 Experimental group B-4 200

Each group was cultivated in an incubator (37° C., 5% CO₂) for 24 hours.The liquid in each well was removed, followed by washing withphosphate-buffered saline (PBS). Thereafter, 100 μL of 95% ethanol wasadded into each well, followed by incubating at room temperature for 10minutes, and the resultant fixed cells were then stained with 100 μL of0.1% crystal violet for 15 minutes. The liquid in each well was removed,followed by washing with PBS. Thereafter, 200 μL of 10% acetic acid wasadded into each well, followed by incubating at room temperature for 10minutes, so as to solubilize crystal violet. The resultant biofilmcontained in each well was subsequently subjected to determination ofabsorbance at a wavelength of 590 nm by a spectrophotometer.

The inhibition rate (%) was calculated using the following Equation(II):

C=(1−D/E)×100  (II)

where C=inhibition rate (%)

-   -   D=OD₅₉₀ value of the respective experimental group or        comparative group    -   E=OD₅₉₀ value of the control group

Results:

Table 5 shows the inhibition rate of each group. It can be seen fromTable 5 that the inhibition rates determined in the experimental groupsL-1 to L-4 were respectively higher than those determined in thecomparative groups L-1 to L-4, and the inhibition rates determined inthe experimental groups B-1 to B-4 were also respectively higher thanthose determined in the comparative groups B-1 to B-4. Therefore, theapplicant contemplates that the postbiotic extract of the presentdisclosure is effective in inhibiting biofilm formation.

TABLE 5 Group Postbiotic extract Inhibition rate (%) Control group —   0% Comparative group L-1 The postbiotic extract of  6.9% Comparativegroup L-2 the prior art from 21.3% Comparative group L-3 Lactobacillusplantarum 40.5% Comparative group L-4 CB102 41.2% Experimental group L-1The postbiotic extract of 10.2% Experimental group L-2 the presentdisclosure from 30.6% Experimental group L-3 Lactobacillus plantarum61.2% Experimental group L-4 CB102 63.2% Comparative group B-1 Thepostbiotic extract of  0.7% Comparative group B-2 the prior art from 8.1% Comparative group B-3 Bifidobacterium longum 19.7% Comparativegroup B-4 CB108 29.1% Experimental group B-1 The postbiotic extract of 1.0% Experimental group B-2 the present disclosure from 10.2%Experimental group B-3 Bifidobacterium longum 27.5% Experimental groupB-4 CB108 36.6%

Example 4. Evaluation for the Effect of Postbiotic Extract According tothis Disclosure on Restoring Healthy Gut Flora Experimental Procedures:

Staphylococcus aureus and a respective one of Lactobacillus plantarumCB102, Lactobacillus acidophilus JCM1132, Lactobacillus casei JCM1134,Bifidobacterium bifidum JCM1255, Bifidobacterium lactis JCM10602,Bifidobacterium longum CB108, and Bacillus coagulans CB106 wereinoculated in a TSB, followed by cultivation in an incubator (37° C., 5%CO₂) for 8 hours. The respective resultant co-culture was divided into 4groups (including one control group, one comparative group, and twoexperimental groups), and each group was treated with a test sample asshown in Table 6.

TABLE 6 Final Co-culture Groups Test sample concentration Co-culture ofControl — — Staphylococcus group 1 aureus and Comparative Inulin 5 g/LLactobacillus group 1 acidophilus Experimental The postbiotic 100 mg/LJCM1132 group 1-L extract of the present disclosure from Lactobacillusplantarum CB102 Experimental The postbiotic 200 mg/L group 1-B extractof the present disclosure from Bifidobacterium longum CB108 Co-cultureof Control — — Staphylococcus group 2 aureus and Comparative Inulin 5g/L Lactobacillus group 2 casei JCM1134 Experimental The postbiotic 100mg/L group 2-L extract of the present disclosure from Lactobacillusplantarum CB102 Experimental The postbiotic 200 mg/L group 2-B extractof the present disclosure from Bifidobacterium longum CB108 Co-cultureof Control — — Staphylococcus group 3 aureus and Comparative Inulin 5g/L Lactobacillus group 3 plantarum CB102 Experimental The postbiotic100 mg/L group 3-L extract of the present disclosure from Lactobacillusplantarum CB102 Experimental The postbiotic 200 mg/L group 3-B extractof the present disclosure from Bifidobacterium longum CB108 Co-cultureof Control — — Staphylococcus group 4 aureus and Comparative Inulin 5g/L Bifidobacterium group 4 lactis JCM10602 Experimental The postbiotic100 mg/L group 4-L extract of the present disclosure from Lactobacillusplantarum CB102 Experimental The postbiotic 200 mg/L group 4-B extractof the present disclosure from Bifidobacterium longum CB108 Co-cultureof Control — — Staphylococcus group 5 aureus and Comparative Inulin 5g/L Bifidobacterium group 5 bifidum JCM1255 Experimental The postbiotic100 mg/L group 5-L extract of the present disclosure from Lactobacillusplantarum CB102 Experimental The postbiotic 200 mg/L group 5-B extractof the present disclosure from Bifidobacterium longum CB108 Co-cultureof Control — — Staphylococcus group 6 aureus and Comparative Inulin 5g/L Bifidobacterium group 6 longum CB108 Experimental The postbiotic 100mg/L group 6-L extract of the present disclosure from Lactobacillusplantarum CB102 Experimental The postbiotic 200 mg/L group 6-B extractof the present disclosure from Bifidobacterium longum CB108 Co-cultureof Control — — Staphylococcus group 7 aureus and Comparative Inulin 5g/L Bacillus group 7 coagulans CB106 Experimental The postbiotic 100mg/L group 7-L extract of the present disclosure from Lactobacillusplantarum CB102 Experimental The postbiotic 200 mg/L group 7-B extractof the present disclosure from Bifidobacterium longum CB108

All the groups were cultivated in an incubator (37° C., 5% CO₂) for 8hours. Thereafter, a suitable amount of the respective resultantco-culture was coated onto a MRS agar plate using spread platetechnique, followed by cultivation in an incubator (37° C., 5% CO₂)overnight. The number of colonies of probiotic microbes on the MRS agarplate of each group was counted, and the log value of the colony formingunit (CFU/mL) was further calculated and the viable cell count wasindicated by log CFU/mL.

Results:

Table 7 shows the viable cell count of probiotic microbes in each group.It can be seen from Table 7 that, for the co-culture of Staphylococcusaureus and Lactobacillus acidophilus JCM1132, the increased viable cellcounts of probiotic microbes (i.e., Lactobacillus acidophilus JCM1132)determined in the experimental groups 1-L and 1-B were higher than thosedetermined in the comparative group 1 and the control group 1. Besides,similar satisfactory results were observed with respect to theexperimental groups 2-L to 7-L and 2-B to 7-B, indicating that thepostbiotic extract of the present disclosure can effectively prevent theimbalance of the gut microbiota, and can eliminate or decrease potentialor known pathogenic populations in the gut or intestine, and hence iscapable of restoring and maintaining a healthy gut microbiota.

TABLE 7 Increased Viable cell Group Test probiotic strain count (logCFU/mL) Control group 1 Lactobacillus 0.8 Comparative group 1acidophilus 1.1 Experimental group 1-L JCM1132 2.0 Experimental group1-B 1.3 Control group 2 Lactobacillus 0.5 Comparative group 2 caseiJCM1134 0.8 Experimental group 2-L 1.7 Experimental group 2-B 1.2Control group 3 Lactobacillus 0.4 Comparative group 3 plantarum CB1020.7 Experimental group 3-L 1.7 Experimental group 3-B 1.2 Control group4 Bifidobacterium 0.3 Comparative group 4 lactis JCM10602 0.6Experimental group 4-L 1.8 Experimental group 4-B 1.2 Control group 5Bifidobacterium 0.1 Comparative group 5 bifidum JCM1255 0.2 Experimentalgroup 5-L 0.8 Experimental group 5-B 0.5 Control group 6 Bifidobacterium0.1 Comparative group 6 longum CB108 0.3 Experimental group 6-L 0.5Experimental group 6-B 0.4 Control group 7 Bacillus coagulans 0.2Comparative group 7 CB106 0.2 Experimental group 7-L 0.6 Experimentalgroup 7-B 0.4

Example 5. Evaluation for the Effect of Postbiotic Extract According tothis Disclosure on Modulating Gut Immunity A. Effect of PostbioticExtract on TGF-β Content of Caco-2 Cells

Caco-2 cells were divided into 25 groups, including one control group,twelve comparative groups (i.e., comparative groups L-1 to L-6 and B-1to B-6), and twelve experimental groups (i.e., experimental groups L-1to L-6 and B-1 to B-6). Each group of the Caco-2 cells was incubated ina respective well of a 96-well culture plate containing 200 μL of DMEMat 1×10⁴ cells/well, followed by cultivation in an incubator (37° C., 5%CO₂) for 24 hours. After medium change with a fresh medium, each of thecell cultures of the twelve comparative groups and twelve experimentalgroups was treated with the respective test postbiotic extract so thatthe cell culture of each group had a final concentration of therespective test postbiotic extract as shown in Table 8. The cell cultureof the control group received no treatment.

TABLE 8 Group Test postbiotic extract Final concentration (mg/L) Controlgroup — 0 Comparative group L-1 The postbiotic 2.5 Comparative group L-2extract of the 5 Comparative group L-3 prior art from 25 Comparativegroup L-4 Lactobacillus 50 Comparative group L-5 plantarum CB102 100Comparative group L-6 200 Experimental group L-1 The postbiotic 2.5Experimental group L-2 extract of the 5 Experimental group L-3 present25 Experimental group L-4 disclosure from 50 Experimental group L-5Lactobacillus 100 Experimental group L-6 plantarum CB102 200 Comparativegroup B-1 The postbiotic 2.5 Comparative group B-2 extract of the 5Comparative group B-3 prior art from 25 Comparative group B-4Bifidobacterium 50 Comparative group B-5 longum CB108 100 Comparativegroup B-6 200 Experimental group B-1 The postbiotic 2.5 Experimentalgroup B-2 extract of the 5 Experimental group B-3 present 25Experimental group B-4 disclosure from 50 Experimental group B-5Bifidobacterium 100 Experimental group B-6 longum CB108 200

After cultivation in an incubator (37° C., 5% CO₂) for 24 hours, therespective resultant cell culture was subjected to the determination ofTGF-β content according to the method described in section 1 of “GeneralProcedures”.

As shown in Table 9 below, the TGF-β contents determined in theexperimental groups L-1 to L-6 were respectively higher than thosedetermined in the comparative groups L-1 to L-6, and the TGF-β contentsdetermined in the experimental groups B-1 to B-6 were also respectivelyhigher than those determined in the comparative groups B-1 to B-6. Thisresult suggests that the postbiotic extract of the present disclosure iseffective in inducing TGF-β secretion in Caco-2 cells.

TABLE 9 Group TGF-ß content (pg/mL) Control group 5.1 Comparative groupL-1 10.4 Comparative group L-2 25.6 Comparative group L-3 42.6Comparative group L-4 56.4 Comparative group L-5 58.5 Comparative groupL-6 58.7 Experimental group L-1 20.8 Experimental group L-2 48.5Experimental group L-3 81.2 Experimental group L-4 102.0 Experimentalgroup L-5 103.5 Experimental group L-6 103.6 Comparative group B-1 10.1Comparative group B-2 10.2 Comparative group B-3 37.4 Comparative groupB-4 59.1 Comparative group B-5 76.8 Comparative group B-6 77.6Experimental group B-1 20.7 Experimental group B-2 20.8 Experimentalgroup B-3 75.2 Experimental group B-4 119.8 Experimental group B-5 152.5Experimental group B-6 153.9

B. Effect of Postbiotic Extract on TGF-β Content of Caco-2 Cells in thePresence of Gastric Acid

A suitable amount of a respective one of Lactobacillus plantarum CB102,Lactobacillus acidophilus JCM1132, Lactobacillus casei JCM1134,Bifidobacterium bifidum JCM1255, Bifidobacterium lactis JCM10602,Bifidobacterium longum CB108, the postbiotic extract of the presentdisclosure from Lactobacillus plantarum CB102, and the postbioticextract of the present disclosure from Bifidobacterium longum CB108 wasmixed with artificial gastric acid (containing 0.137 M NaCl, 0.0027 MKCl, 0.01 M Na₂HPO₄, and 0.0018 M NaH₂PO₄, and having a pH of 2),followed by cultivation in an incubator (37° C., 5% CO₂) for 3 hours.The resultant mixtures were used for the following experiment and arereferred to as “gastric acid-treated” test samples hereinafter.

In addition, Caco-2 cells were divided into 16 groups, including eightcomparative groups (i.e., comparative groups 1-8) and eight experimentalgroups (i.e., experimental groups 1-8). Each group of the Caco-2 cellswas incubated in a respective well of a 96-well culture plate containing200 μL of DMEM at 1×10⁴ cells/well, followed by cultivation in anincubator (37° C., 5% CO₂) for 24 hours. After medium change with afresh medium, each of the cell cultures of the eight comparative groupsand eight experimental groups was treated with the respective testsample so that the cell culture of each group had a final concentrationof the respective test sample as shown in Table 10.

TABLE 10 Group Test sample Final concentration Comparative group 1Lactobacillus 1 × 10¹⁰ CFU/L acidophilus JCM1132 Experimental group 1Gastric acid-treated Lactobacillus acidophilus JCM1132 Comparative group2 Lactobacillus casei JCM1134 Experimental group 2 Gastric acid-treatedLactobacillus casei JCM1134 Comparative group 3 Lactobacillus plantarumCB102 Experimental group 3 Gastric acid-treated Lactobacillus plantarumCB102 Comparative group 4 Bifidobacterium lactis JCM10602 Experimentalgroup 4 Gastric acid-treated Bifidobacterium lactis JCM10602 Comparativegroup 5 Bifidobacterium bifidum JCM1255 Experimental group 5 Gastricacid-treated Bifidobacterium bifidum JCM1255 Comparative group 6Bifidobacterium longum CB108 Experimental group 6 Gastric acid-treatedBifidobacterium longum CB108 Comparative group 7 The postbiotic 100 mg/Lextract of the present dsiclosure from Lactobacillus plantarum CB102Experimental group 7 Gastric acid-treated postbiotic extract of thepresent disclosure from Lactobacillus plantarum CB102 Comparative group8 The postbiotic extract of the present disclosure from Bifidobacteriumlongum CB108 Experimental group 8 Gastric acid-treated postbioticextract of the present disclosure from Bifidobacterium longum CB108

After cultivation in an incubator (37° C., 5% CO₂) for 24 hours, therespective resultant cell culture was subjected to the determination ofTGF-β content according to the method described in section 1 of “GeneralProcedures”.

As shown in Table 11 below, the TGF-β contents determined in theexperimental groups 1-6 were significantly lower than those determinedin the comparative groups 1-6, respectively. However, the TGF-β contentsof the experimental groups 7-8 were only slightly decreased as comparedto the comparative groups 7-8, respectively, indicating that thepostbiotic extract of the present disclosure has an excellent acidtolerance and is able to overcome the environmental pressure posed bythe human digestive tract, and hence can reach the intestine (s) andcolonize the probiotics therein after ingestion.

TABLE 11 Group TGF-ß content (pg/mL) Comparative group 1 62.2Experimental group 1 20.1 Comparative group 2 112.1 Experimental group 285.9 Comparative group 3 40.6 Experimental group 3 31.1 Comparativegroup 4 101.1 Experimental group 4 23.6 Comparative group 5 143.6Experimental group 5 53.4 Comparative group 6 131.7 Experimental group 642.2 Comparative group 7 103.5 Experimental group 7 85.5 Comparativegroup 8 154.2 Experimental group 8 140.3C. Effect of the Combination of Postbiotic Extract with Probiotics onTGF-β Content of Caco-2 Cells

Caco-2 cells were divided into 21 groups, including seven comparativegroups (i.e., comparative groups 1-7) and fourteen experimental groups(i.e., experimental groups 1-L to 7-L and 1-B to 7-B). Each group of theCaco-2 cells was incubated in a respective well of a 96-well cultureplate containing 200 μL of DMEM at 1×10⁴ cells/well, followed bycultivation in an incubator (37° C., 5% CO₂) for 24 hours. After mediumchange with a fresh medium, the culture of each group was treated withprobiotics only or additionally with the postbiotic extract of thepresent disclosure so that the cell culture of each group respectivelyhad final concentrations of the probiotics only or additionally with thepostbiotic extract as shown in Table 12.

TABLE 12 The postbiotic The postbiotic extract of the extract of thepresent disclosure present disclosure from from LactobacillusBifidobacterium Probiotic plantarum longum CB108 Group (1 × 10¹⁰ CFU/L)CB102 (100 mg/L) (100 mg/L) Comparative Lactobacillus − − group 1acidophilus Experimental JCM1132 + − group 1-L Experimental − + group1-B Comparative Lactobacillus − − group 2 casei JCM1134 Experimental + −group 2-L Experimental − + group 2-B Comparative Lactobacillus − − group3 plantarum Experimental CB102 + − group 3-L Experimental − + group 3-BComparative Bifidobacterium − − group 4 lactis JCM10602 Experimental + −group 4-L Experimental − + group 4-B Comparative Bifidobacterium − −group 5 bifidum JCM1255 Experimental + − group 5-L Experimental − +group 5-B Comparative Bifidobacterium − − group 6 longum CB108Experimental + − group 6-L Experimental − + group 6-B ComparativeBacillus − − group 7 coagulans Experimental CB106 + − group 7-LExperimental − + group 7-B

After cultivation in an incubator (37° C., 5% CO₂) for 24 hours, therespective resultant cell culture was subjected to the determination ofTGF-β content according to the method described in section 1 of “GeneralProcedures”.

As shown in Table 13 below, the TGF-β contents determined in theexperimental groups 1-L to 7-L and 1-B to 7-B were respectively higherthan those determined in the comparative groups 1-7, indicating that thecombination of a postbiotic extract with probiotics is effective ininducing TGF-β secretion in Caco-2 cells.

TABLE 13 Group TGF-ß content (pg/mL) Comparative group 1 20.1Experimental group 1-L 100.7 Experimental group 1-B 155.7 Comparativegroup 2 85.9 Experimental group 2-L 152.0 Experimental group 2-B 207.0Comparative group 3 31.1 Experimental group 3-L 109.3 Experimental group3-B 164.3 Comparative group 4 23.6 Experimental group 4-L 103.4Experimental group 4-B 158.4 Comparative group 5 53.4 Experimental group5-L 126.7 Experimental group 5-B 181.7 Comparative group 6 42.2Experimental group 6-L 117.9 Experimental group 6-B 172.9 Comparativegroup 7 52.5 Experimental group 7-L 126.0 Experimental group 7-B 181.0

Summarizing the above test results, it is clear that the postbioticextract of the present disclosure has an excellent acid tolerance, andcan effectively induce TGF-β secretion in Caco-2 cells, and hence can beused as a food additive for modulating gut immunity.

All patents and references cited in this specification are incorporatedherein in their entirety as reference. Where there is conflict, thedescriptions in this case, including the definitions, shall prevail.

While the disclosure has been described in connection with what areconsidered the exemplary embodiments, it is understood that thisdisclosure is not limited to the disclosed embodiments but is intendedto cover various arrangements included within the spirit and scope ofthe broadest interpretation so as to encompass all such modificationsand equivalent arrangements.

What is claimed is:
 1. A method of inhibiting biofilm formation,comprising administering to a subject in need thereof a postbioticextract, wherein the postbiotic extract is prepared by a processincluding the steps of: a) providing a whey protein having anisoelectric point of 4.4 and a dextrin having an isoelectric point of5.2; b) admixing the whey protein and a probiotic bacterium with waterhaving a pH value of 7.5, so as to form a mixture, wherein the probioticbacterium is selected from the group consisting of Lactobacillusplantarum CB102, Lactobacillus acidophilus JCM1132, Lactobacillus caseiJCM1134, Bifidobacterium bifidum JCM1255, Bifidobacterium lactisJCM10602, Bifidobacterium longum CB108, and combinations thereof; c)adding the dextrin into the mixture, followed by adjusting the pH valueof the dextrin-added mixture to between 4.4 and 5.2, so that aprecipitate is formed; and d) subjecting the precipitate to an acidhydrolysis treatment with lactic acid to isolate the cell wall of theprobiotic bacterium, so as to obtain the postbiotic extract; wherein thebiofilm formation is caused by Staphylococcus aureus.